Role of Hypoxia in the Progression and Dissemination of Multiple Myeloma.

Multiple myeloma (MM) is characterized by the disseminated involvement of the bone-marrow (BM), and its progression involves a continuous circulation of the MM cells in the peripheral blood and homing back to the BM. Several reports have described the mechanism involved in homing of MM cells to the BM. However, the driving force to metastasize from one site of the BM to another is not yet understood. Hypoxia (oxygen deprivation) was associated with poor patient prognosis in solid tumors, and several studies have shown that BM has a hypoxic nature. We hypothesize that the hypoxic nature of the BM and the rapid development of MM may cause induction of hypoxia in the tumor microenvironment, induce stress in the MM cells, and drive them to disseminate to new BM niches with normal oxygen levels (normoxia).

First we have tested the level of oxygenation of MM cells and cells from its BM microenvironment by injection of pimonidazole (PIM) to mice with MM and determined the levels of PIM binding in MM cells and stromal cells in the microenvironment by flow cytometry. The whole cells population in the BM was hypoxic, while the MM cells were more hypoxic compared to cells in the microenvironment. We have mimicked these results in vitro by incubation of MM cells (cell lines and patients samples) in hypoxic conditions (0.5% oxygen, for 24 hrs). Induction of hypoxia was also verified by detection of increased binding of PIM by flow cytometry, and increased levels of HIF1 and HIF2 in MM cells by immunoblotting. MTT assay showed that hypoxia significantly decreased (40%) the survival of MM cells. Immunoblotting showed a downregulation of proliferative signaling pathway, including PI3K, AKT, mTOR and ERK; and induction of stress pathways including MKK-3 and 6, and p38. Apoptosis was not detected by flow cytometry after 24 hrs of hypoxic conditions. These results were confirmed by immunoblotting, which showed no change in apoptosis related proteins including caspases 3 and 7, Bcl-2, Bcl-xL and Mcl-1. However, hypoxia induced G1-phase cell cycle arrest (increase of 20% in G1)., These results were confirmed by immunoblotting showing downregulation of proteins associated with G1 to S phase transition including cyclins D1, D2, D3 and E1, and pRb; and upregulation of cell cycle inhibitor p27. Testing the effect of hypoxia on the adhesion properties of MM cells to BM stromal cells (BMSCs) revealed that hypoxic MM cells were 50% less adherent to BMSCs compared to normoxic MM cells. Also, hypoxic BMSCs induced 30% less adhesion of MM cells compared to normoxic BMSCs. Mechanistically, immunoblotting showed a significant decrease in the expression of cadherins in both hypoxic MM cells and hypoxic BMSCs. The chemokine stromal-cell derived factor-1-alpha (SDF1) is known to increase adhesive properties of MM cells, and to induce MM retention in the BM. Testing the levels of SDF1 revealed that hypoxia decreased SDF1 secretion (40%) from BMSCs. As a result, media from hypoxic BMSCs induce significantly decreased MM cell chemotaxis (20%) compared to media from normoxic BMSCs. These findings suggest that hypoxic BM will retain less MM cells in the BM and allow more egression. Comparison of the ability of hypoxic and normoxic MM cells to migrate towards normoxic BMSCs or SDF1 revealed that hypoxic MM cells had increased (250% and 350%, respectively) chemotactic properties compared to normoxic MM cells. These findings correlated with the increased expression of CXCR4 on hypoxic MM cells (mean fluorescent intensity shifts: Isotype=90, normoxic = 520, hypoxic = 1100). These results suggest that hypoxic MM cells will home to normoxic BM niches more efficiently than normoxic MM cells. Finally, testing the recovery of the hypoxic MM cells after incubation in normoxic conditions showed that the expression of CXCR4 was completely downregulated to levels found in normoxic cells after 6 hrs of exposure to normoxia. Moreover, incubation of hypoxic MM cells in normoxic BMSCs- media or SDF1 restored their expression of cadherins and their ability to adhere to BMSCs.

In conclusion, we have showed that hypoxia inhibited the proliferation of MM cells and decreased their adhesion to BMSCs, as a first step of egress and metastasize to new normoxic BM niches.

Anderson:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Ghobrial:Millennium: Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau.